PEG-DSPE Block Copolymers and Their Derivatives

Polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) block copolymer is a biocompatible and amphiphilic polymer, which can be widely used in the preparation of liposomes, polymer nanoparticles, polymerization hybrid nanoparticles, solid lipid nanoparticles, lipid-polymer hybrid nanoparticles and microemulsions. In particular, the terminal groups of PEG can be activated and connected to various targeted ligands, which can prolong the drug circulation time, improve the bioavailability of drugs, and reduce adverse reactions, especially for specific cells, tissues, and even intracellular localization in organelles.

Fig. 1. The application of DSPE-PEG-Mal in nanoparticles (Mol. Pharmaceutics. 2021, 18(9): 3235-3246).

What is PEG-DSPE?

PEG-DSPE amphoteric polymer is a nanostructure composed of a hydrophobic core (DSPE) and a hydrophilic shell (PEG). The core-shell structure can encapsulate and carry poorly soluble drugs to accumulate in the core of DSPE, while the PEG shell can reduce the in vivo clearance of cholesterol-free liposomes and adsorption to plasma proteins. Therefore, using PEG-DSPE to form nanostructures can prolong the circulation time in the human body and sustain the release of drugs within the optimal drug concentration range. In addition, the content of PEG-DSPE has a great influence on the size of microcapsules. Sriwongsitanont and Ueno used a decontamination method to prepare egg yolk phosphatidylcholine microcapsules containing DSPE-PEG 2000, and confirmed that the addition of DSPE-PEG 2000 promoted the formation of microcapsules. As the PEG lipid content increases, the size of egg yolk phosphatidylcholine microcapsules decreases.

Synthesis of PEG-DSPE Derivatives

A commonly used lipid derivative of PEG is methoxy-PEG-DSPE with a methoxy terminus. Although methoxy-PEG-DSPE can extend the circulation time of liposomes, the methoxy groups are too inactive to react with ligands under mild conditions. Therefore, it is necessary to modify PEG-DSPE with terminal groups mainly composed of hydroxyl groups in order to connect with certain ligands. By modifying the hydroxyl groups of the PEG end groups, the physical and chemical properties of the polymer are improved. Common terminal group forms of PEG-DSPE derivatives mainly include carboxylation, amination and acylation.

Carboxy-PEG-DSPE (DSPE-PEG-COOH)

Carboxyl groups are introduced into the end groups of the PEG-DSPE block copolymer, which can easily interact with active target cells or tissue ligands, such as transporters and peptides. Some reports have successfully synthesized DSPE-PEG-COOH. Briefly, DSPE was added to polyethylene glycol bis(succinimidyl succinate) (PEG-2OSu) in chloroform methanol, followed by triethylamine. The reaction mixture was stirred vigorously at room temperature overnight. After the product was separated by thin layer chromatography, the complete conversion of the primary amino groups of DSPE was confirmed by the negative reactivity of ninhydrin. Vaidy et al. used DSPE-PEG-COOH to couple with c(RGD) amine group to prepare arginine-glycine-aspartic acid (RGD) coupled liposomes (CNPRGDY[OET]RC). Compared with ordinary streptokinase solution and long-circulating liposomes, RGD peptide-conjugated liposomes accumulate at the thrombus and increase thrombolytic activity.

Amino-PEG-DSPE (DSPE-PEG-NH2)

The amino groups of heterobifunctional PEG can be selectively protected by protecting groups such as fluormethoxycarbonyl and butoxycarbonyl (Boc). The other end of PEG is an active group that reacts with DSPE. The protecting groups are then removed after the reaction to form amino-PEG-DSPE. In addition, amino-PEG-DSPE can also be combined with small molecule drugs and ligands. The following amino-PEG-DSPE was synthesized by Zalipsky et al. First, the amino group of heterobifunctional PEG is selectively protected by the Boc group. Secondly, a succinimide carbonate (SC) group was introduced at the hydroxyl end of α-Boc-ω-hydroxy-PEG to connect the amino group of DSPE with the PEG primary amine functional group after the BOC group was removed by acid.

Fig. 2. Synthesis of amino-PEG-DSPE.

Hydrazide-PEG-DSPE

The ligand can also be covalently combined with the hydrazide group attached to PEG-DSPE to form a hydrazide bond. It is reported that the oxidized ligand reacts with the hydrazide group on PEG-DSPE to form Hydrazide-PEG-DSPE. Zalipsky prepared heterobifunctional PEG derivatives by modifying one end with an SC group and protecting the hydrazide group with Tert-Boc on the other end. Then the amino groups of Boc-PEG-SC and DSPE react easily to produce Boc-PEG-DSPE. Finally, the Boc group was removed by acidolysis to obtain Hydrazide-PEG-DSPE conjugates suitable for connecting various ligands. The above method has been successfully used to synthesize various ligand-modified PEG-DSPE copolymers for targeted drug delivery systems.

Maleimide-PEG-DSPE (Mal-PEG-DSPE)

In recent years, PEG-DSPE modified with a maleimide group at the end of the PEG chain has been widely used in targeted delivery systems due to its convenient and rapid reaction with ligands such as antibodies and peptides. Two methods have been successfully adopted to synthesize Mal-PEG-DSPE. In the first method, amino-PEG-DSPE is reacted with N-succinimide 3-(N-maleimide)-propionate in CHCl, DMF, and triethylamine to form Mal-PEG-DSPE. In the second method, ω-(β-[N-maleimido])-PEG-α-succinimide carboxylate (Mal-PEG-SC) was synthesized with DSPE and triethylamine in chloroform.

Fig. 3. Synthesis of Mal-PEG-DSPE.

Targeted Drug Delivery of PEG-DSPE

Targeted drug delivery systems are considered a promising strategy to improve the selective targeting of drugs to diseased tissues, thereby improving therapeutic efficacy and reducing drug toxicity. In particular, various targeting groups such as antibodies, growth factors or cytokine functions are used to introduce drugs, proteins and nucleic acids into target cells. The terminal groups of PEG-DSPE are easily grafted to different targeting groups. These targeting groups are mainly divided into non-antibody ligand targeting groups and antibody targeting groups.

Non-antibody Targeting Molecules

Due to changes in the microenvironment, some cells or tissues express high levels of specific receptors, while normal tissues express lower levels or undetectable receptors. Therefore, it is possible to deliver drugs to target sites by targeting specific receptors through ligands attached to the termini of PEG-DSPE. For example, experimental non-targeting PEG-terminated liposomes in tumors showed that their distribution was restricted to extracellular fluid and tumor-infiltrating macrophages, but folate (FA)-targeted liposomes delivered the drug to folate-expressing receptor (FR) in tumor cells, which has a better antitumor effect than limited, gradual distribution into the extracellular fluid.

Table 1. Non-antibody targeting molecules attached to PEG-DSPE for cancer therapy.

Non-antibody ligands are often readily available, cheap to manufacture, and easy to handle, and their adverse effects stem primarily from their relatively nonselective expression. Therefore, it is necessary to select appropriate methods and targeting ligands to modify PEG-DSPE end groups. So far, many non-antibody targeting molecules (Table 1) have been attached to PEG-DSPE for cancer treatment.

Antibody Targeting Molecules

With the development of antibody engineering and phage display technology, antibody-mediated targeted therapy has been explored. Based on their broad affinity and small molecule size, these technologies are used to achieve high specificity for target tissues. When appropriate antibodies are linked to the reactive ends of PEG-DSPE, the vector can be targeted to selected tissues, depending on the ability of the antibody or ligand to promote cell-specific docking. For example, anti-CD22 monoclonal antibodies specifically bind to the expression of the CD22 surface antigen in non-Hodgkin lymphoma cells. The post-insertion method was used to introduce anti-CD22-PEG-DSPE into liposomes containing anticancer drugs. Anti-CD22 immunoliposomes have higher efficacy and lower toxicity than unmodified CD22 immunoliposomes.

Table 2. Antibody targeting molecules grafted onto PEG-DSPE for targeted delivery.

The binding of immunoliposomes (anti-CD19) to human CD19+ B lymphoma cell line (Namalwa) is 3 times higher than that of non-targeting liposomes. The ability of non-targeting liposomes to recognize B cells or T cells is significantly lower than that of targeted DXR immunoliposomes (anti-CD19). Many antibodies targeting molecules listed in Table 2 have been studied for targeted delivery by grafting onto the terminal groups of PEG-DSPE.

Molecular therapies, including gene therapy, are promising strategies for treating human diseases. However, efficient and specific delivery of molecular therapeutics to target tissues remains a significant challenge. Fortunately, researchers have discovered that in vivo tumor-targeted therapy can be achieved by encapsulating genes into nanocarriers due to their enhanced permeability and retention effects. In addition, the end groups of PEG can be activated and linked to various targeting ligands on the nanocarrier surface, which has been shown to improve delivery efficiency and tissue specificity. Please refer to drug carriers to learn about the use of PEG-DSPE in drug delivery.

Reference

1. Neves, N.M. et al. Cellular uptake of three different nanoparticles in an inflammatory arthritis scenario versus normal conditions. Mol. Pharmaceutics. 2021, 18(9): 3235-3246.